This invention is related to the field of environmental toxicology, in particular to methods for measuring the effects of environmental toxins.
The human mitochondrial (mt) genome is small (16.5 kb) and encodes 13 respiratory chain subunits, 22 tRNAs and two rRNAs. Mitochondrial DNA is present at extremely high levels (103-104 copies per cell) and the vast majority of these copies are identical (homoplasmic) at birth (1). Expression of the entire complement of mt genes is required to maintain proper function of the organelle, suggesting that even slight alterations in DNA sequences could have profound effects (2). It is generally accepted that mtDNA mutations are generated endogenously during oxidative phosphorylation via pathways involving reactive oxygen species (ROS), but they can also be generated by external carcinogens or environmental toxins. These mutations may accumulate partially because mitochondria lack protective histones and highly efficient DNA repair mechanisms as seen in the nucleus (3). Recently several mtDNA mutations were found specifically in human colorectal cancer (4).
It is an object of the present invention to provide methods of monitoring exposure of a person to an environmental pollutant.
It is another object of the present invention to provide a kit for monitoring exposure of a person to environmental pollutants.
It is an object of the invention to provide methods to aid in the detection of cancer or metastasis.
It is an object of the invention to provide probes and primers for detecting mitochondrial mutations.
It is an object of the invention to provide a method to aid in detecting the presence of tumor cells in a patient.
These and other objects of the invention are achieved by providing one or more of the embodiments described below. In one embodiment a method is provided for monitoring exposure of a person to an environmental pollutant. The presence of one or more mutations in mitochondrial DNA (mtDNA) in a body fluid of a person exposed to an environmental pollutant is determined at two or more time points. The amounts of mutations in mtDNA at different time points are compared. The amount of mutations correlates with amount of exposure to the environmental pollutant.
According to another embodiment another method is provided for monitoring exposure of a person to an environmental pollutant. The prevalence of one or more mutations in mitochondrial DNA (mtDNA) in a body fluid of a person exposed to an environmental pollutant is measured. A measured prevalence of one or more mutations in mtDNA of greater than 1% indicates clonal expansion of cells which harbor the one or more mutations in the person.
According to still another embodiment of the invention a method is provided for monitoring exposure of a person to an environmental pollutant. One or more mutations in a D-loop of mitochondrial DNA (mtDNA) in a body fluid of a person exposed to an environmental pollutant are measured. The number of mutations in mtDNA correlates with exposure to the environmental pollutant.
According to yet another embodiment of the invention a kit is provided. The kit comprises one or more primers which hybridize to a mitochondrial D-loop for making a primer extension product. In addition, the kit contains written material identifying mutations which are found in the D-loop as a result of exposure to one or more environmental pollutants.
According to another embodiment of the invention an oligonucleotide probe is provided. The probe comprises a sequence of at least 10 contiguous nucleotides of a human mitochondrial genome. The probe can optionally contain at least 12, 14, 16, 18, 20, 22, 24, 26, or 30 such contiguous nucleotides. The oligonucleotide comprises a mutation selected from the group consisting of: a mutation selected from the group consisting of: Txe2x86x92C at nucleotide 114; xcex94C at nucleotide 302; Cxe2x86x92A at nucleotide 386; insert T at nucleotide 16189; Axe2x86x92C at nucleotide 16265; Axe2x86x92T at nucleotide 16532; Cxe2x86x92T at nucleotide 150; Txe2x86x92C at nucleotide 195; xcex94C at nucleotide 302; Cxe2x86x92A at nucleotide 16183; Cxe2x86x92T at nucleotide 16187; Txe2x86x92C at nucleotide 16519; Gxe2x86x92A at nucleotide 16380; Gxe2x86x92A at nucleotide 75; insert C at nucleotide 302; insert CG at nucleotide 514; Txe2x86x92C at nucleotide 16172; Cxe2x86x92T at nucleotide 16292; Axe2x86x92G at nucleotide 16300; Axe2x86x92G at nucleotide 10792; Cxe2x86x92T at nucleotide 10793; Cxe2x86x92T at nucleotide 10822; Axe2x86x92G at nucleotide 10978; Axe2x86x92G at nucleotide 11065; Gxe2x86x92A at nucleotide 11518; Cxe2x86x92T at nucleotide 12049; Txe2x86x92C at nucleotide 10966; Gxe2x86x92A at nucleotide 11150; Gxe2x86x92A at nucleotide 2056; Txe2x86x92C at nucleotide 2445; Txe2x86x92C at nucleotide 2664; Txe2x86x92C at nucleotide 10071; Txe2x86x92C at nucleotide 10321; Txe2x86x92C at nucleotide 12519; xcex94 7 amino acids at nucleotide 15642; Gxe2x86x92A at nucleotide 5521; Gxe2x86x92A at nucleotide 12345; Txe2x86x92C substitution at position 710; Txe2x86x92C substitution at position 1738; Txe2x86x92C substitution at position 3308; Gxe2x86x92A substitution at position 8009; Gxe2x86x92A substitution at position 14985; Txe2x86x92C substitution at position 15572; Gxe2x86x92A substitution at position 9949; Txe2x86x92C substitution at position 10563; Gxe2x86x92A substitution at position 6264; A insertion at position 12418; Txe2x86x92C substitution at position 1967; Txe2x86x92A substitution at position 2299; and Gxe2x86x92A at nucleotide 3054.
According to another aspect of the invention an oligonucleotide primer is provided. It comprises a sequence of at least 10 contiguous nucleotides of a human mitochondrial genome. The primer can optionally contain at least 12, 14, 16, 18, 20, 22, 24, 26, or 30 such contiguous nucleotides. The oligonucleotide comprises a mutation selected from the group consisting of: a mutation selected from the group consisting of: Txe2x86x92C at nucleotide 114; xcex94C at nucleotide 302; Cxe2x86x92A at nucleotide 386; insert T at nucleotide 16189; Axe2x86x92C at nucleotide 16265; Axe2x86x92T at nucleotide 16532; Cxe2x86x92T at nucleotide 150; Txe2x86x92C at nucleotide 195; xcex94C at nucleotide 302; Cxe2x86x92A at nucleotide 16183; Cxe2x86x92T at nucleotide 16187; Txe2x86x92C at nucleotide 16519; Gxe2x86x92A at nucleotide 16380; Gxe2x86x92A at nucleotide 75; insert C at nucleotide 302; insert CG at nucleotide 514; Txe2x86x92C at nucleotide 16172; Cxe2x86x92T at nucleotide 16292; Axe2x86x92G at nucleotide 16300; Axe2x86x92G at nucleotide 10792; Cxe2x86x92T at nucleotide 10793; Cxe2x86x92T at nucleotide 10822; Axe2x86x92G at nucleotide 10978; Axe2x86x92G at nucleotide 11065; Gxe2x86x92A at nucleotide 11518; Cxe2x86x92T at nucleotide 12049; Txe2x86x92C at nucleotide 10966; Gxe2x86x92A at nucleotide 11150; Gxe2x86x92A at nucleotide 2056; Txe2x86x92C at nucleotide 2445; Txe2x86x92C at nucleotide 2664; Txe2x86x92C at nucleotide 10071; Txe2x86x92C at nucleotide 10321; Txe2x86x92C at nucleotide 12519; xcex94 7 amino acids at nucleotide 15642; Gxe2x86x92A at nucleotide 5521; Gxe2x86x92A at nucleotide 12345; Txe2x86x92C substitution at position 710; Txe2x86x92C substitution at position 1738; Txe2x86x92C substitution at position 3308; Gxe2x86x92A substitution at position 8009; Gxe2x86x92A substitution at position 14985; Txe2x86x92C substitution at position 15572; Gxe2x86x92A substitution at position 9949; Txe2x86x92C substitution at position 10563; Gxe2x86x92A substitution at position 6264; A insertion at position 12418; Txe2x86x92C substitution at position 1967; Txe2x86x92A substitution at position 2299; and Gxe2x86x92A at nucleotide 3054.
Another aspect of the invention is a method to aid in detecting the presence of tumor cells in a patient. The presence of a single basepair mutation is detected in a mitochondrial genome of a cell sample of a patient. The mutation is found in a tumor of the patient but not in normal tissue of the patient. The tumor is not a colorectal tumor. The patient is identified as having a tumor if one or more single basepair mutations are determined in the mitochondrial genome of the cell sample of the patient.
Yet another embodiment of the invention is provided by another method to aid in detecting the presence of tumor cells in a patient. The presence of a mutation is determined in a D-loop of a mitochondrial genome of a cell sample of a patient. The mutation is found in a tumor of the patient but not in normal tissue of the patient. The patient is identified as having a tumor if one or more single basepair mutations are determined in the mitochondrial genome of the cell sample of the patient.
According to still another aspect of the invention a method is provided to aid in detecting the presence of tumor cells in a patient. The presence of a single basepair mutation is determined in a mitochondrial genome of a cell sample of a patient. The mutation is found in a cancer of the patient but not in normal tissue of the patient. The cancer is selected from the group of cancers consisting of: lung, head and neck, bladder, brain, breast, lymphoma, leukaemia, skin, prostate, stomach, pancreas, liver, ovarian, uterine, testicular, and bone. The patient is identified as having a tumor if one or more single basepair mutations are determined in the mitochondrial genome of the cell sample of the patient.
According to still another aspect of the invention a method is provided to aid in detecting the presence of tumor cells in a patient. The presence of a single basepair mutation is determined in a mitochondrial genome of a cell sample of a patient. The mutation is found in a tumor of the patient but not in normal tissue of the patient. The cancer is selected from the group of cancers consisting of: lung, head and neck, and bladder. The patient is identified as having a tumor if one or more single basepair mutations are determined in the mitochondrial genome of the cell sample of the patient.
Another embodiment of the invention provides a method to aid in detecting the presence of tumor cells in a patient. The presence of a mutation in a mitochondrial genome of a cell sample of a patient is determined. The mutation is selected from the group consisting of: Txe2x86x92C at nucleotide 114; xcex94C at nucleotide 302; Cxe2x86x92A at nucleotide 386; insert T at nucleotide 16189; Axe2x86x92C at nucleotide 16265; Axe2x86x92T at nucleotide 16532; Cxe2x86x92T at nucleotide 150; Txe2x86x92C at nucleotide 195; xcex94C at nucleotide 302; Cxe2x86x92A at nucleotide 16183; Cxe2x86x92T at nucleotide 16187; Txe2x86x92C at nucleotide 16519; Gxe2x86x92A at nucleotide 16380; Gxe2x86x92A at nucleotide 75; insert C at nucleotide 302; insert CG at nucleotide 514; Txe2x86x92C at nucleotide 16172; Cxe2x86x92T at nucleotide 16292; Axe2x86x92G at nucleotide 16300; Axe2x86x92G at nucleotide 10792; Cxe2x86x92T at nucleotide 10793; Cxe2x86x92T at nucleotide 10822; Axe2x86x92G at nucleotide 10978; Axe2x86x92G at nucleotide 11065; Gxe2x86x92A at nucleotide 11518; Cxe2x86x92T at nucleotide 12049; Txe2x86x92C at nucleotide 10966; Gxe2x86x92A at nucleotide 11150; Gxe2x86x92A at nucleotide 2056; Txe2x86x92C at nucleotide 2445; Txe2x86x92C at nucleotide 2664; Txe2x86x92C at nucleotide 10071; Txe2x86x92C at nucleotide 10321; Txe2x86x92C at nucleotide 12519; xcex94 7 amino acids at nucleotide 15642; Gxe2x86x92A at nucleotide 5521; Gxe2x86x92A at nucleotide 12345; Txe2x86x92C substitution at position 710; Txe2x86x92C substitution at position 1738; Txe2x86x92C substitution at position 3308; Gxe2x86x92A substitution at position 8009; Gxe2x86x92A substitution at position 14985; Txe2x86x92C substitution at position 15572; Gxe2x86x92A substitution at position 9949; Txe2x86x92C substitution at position 10563; Gxe2x86x92A substitution at position 6264; A insertion at position 12418; Txe2x86x92C substitution at position 1967; Txe2x86x92A substitution at position 2299; and Gxe2x86x92A at nucleotide 3054. The patient is identified as having a tumor if one or more mutations are determined in the mitochondrial genome of the cell sample of the patient.
These and other embodiments provide the art with non-invasive tools for monitoring exposure to and the effects of environmental pollutants on the human body as well as early detection methods for cancer and metastasis.